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WO1997006817A1 - Reprise de l'hematopoiese - Google Patents

Reprise de l'hematopoiese Download PDF

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Publication number
WO1997006817A1
WO1997006817A1 PCT/GB1996/002006 GB9602006W WO9706817A1 WO 1997006817 A1 WO1997006817 A1 WO 1997006817A1 GB 9602006 W GB9602006 W GB 9602006W WO 9706817 A1 WO9706817 A1 WO 9706817A1
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Prior art keywords
stem cell
recovery
haematopoietic
cfu
myelosuppressive
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PCT/GB1996/002006
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English (en)
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Brian Iles Lord
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British Biotech Pharmaceuticals Limited
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Priority to AU67501/96A priority Critical patent/AU6750196A/en
Publication of WO1997006817A1 publication Critical patent/WO1997006817A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2053—IL-8

Definitions

  • This invention relates to the use of proteinaceous molecules which are stem cell chemokines (SCCs), e.g. LD78, to regulate the recovery of haematopoiesis after myelosuppressive insult or therapy.
  • SCCs stem cell chemokines
  • continuous administration of an SCC enhances the rate and quality of the recovery of haematopoiesis following cytotoxic myelosuppressive insult or therapy.
  • haematopoietic stem cells are both pluripotential - that is they can give rise to all cell types - and capable of self-renewal. This is defined by their ability to repopulate animals whose haematopoietic system has been destroyed by radiation. Stem cells represent a very small percentage of bone marrow cells, and are normally quiescent. When stimulated to divide, they give rise to more committed, differentiated daughter cells with greater proliferative potential. The term stem cell is often also applied to these so-called early progenitor cells. Sequential rounds of division and differentiation give rise to an enormous amplification of cell numbers, necessary for the production of mature blood ceils.
  • CSFs Colony Stimulating Factors
  • G-CSF granulocyte-colony stimulating factor
  • EPO erythropoietin
  • Leukocytic haematopoietic cells are important in maintaining the body's defence against disease.
  • macrophages and lymphocytes are involved in potentiating the body's response to infection and tumours; granulocytes (neutrophils, eosinophils and basophils) are involved in overcoming infection, parasites and tumours.
  • Other cell types derived from haematopoietic stem cells include platelets and erythrocytes. Platelets form an important element in the haemostatic mechanism through initiating thrombus formation by their adhesion to each other and to damaged surfaces, and by the release of factors which assist in the formation of the fibrin clot. Erythrocytes are mainly involved in the transport of oxygen.
  • neutrophils along with other granulocytes, are an essential component of the body's cellular defences against infection is illustrated by the fact that individuals with a leukocyte dysfunction such as LAD (leukocyte adhesion deficiency) are very prone to infection.
  • LAD leukocyte adhesion deficiency
  • Neutrophils are continuously produced in large numbers from myeloid precursors in the bone marrow. Neutrophils are released into the circulation from where they can enter the tissues in response to chemotactic signals released locally during infection or tissue damage. The activated neutrophil can then attack the infective agent by release of enzymes and free-radicals, as well as by phagocytosis. Circulating and tissue neutrophils have a short half-life of about 2hr.
  • the neutropaenia resulting from chemotherapy or radiotherapy occurs within days of treatment, and leaves the patient vulnerable to infection until the haematopoietic system has recovered sufficiently for neutrophil counts to recover.
  • CSFs such as G-CSF and GM-CSF to enhance the neutrophil recovery rate by stimulating the division and differentiation of neutrophil precursors.
  • G-CSF G-CSF
  • GM-CSF GM-CSF
  • SCIs stem cell inhibitor proteins
  • a known stem cell inhibitor protein known as MIP-1 ⁇ (macrophage inflammatory protein), or huMIP-1 ⁇ or LD78 (for the human form), is a peptide of about 69 amino acids and is a member of a growing family of molecules with homologous structure - the chemokine or intercrine family. Other notable members of this family include IL-8 and platelet factor 4.
  • MIP-1 ⁇ A clinical use for MIP-1 ⁇ emerged when it was discovered that it was the same molecule as a factor purified from bone marrow some years earlier (3).
  • This factor stem cell inhibitor protein, was defined by its ability to put early haematopoietic progenitor cells (stem cells) out of cycle. Because stem cells are needed for repopulation of the bone marrow, there is a great deal of interest in the use of this protein (also known as LD78) as a marrow protective agent during cancer chemotherapy.
  • LD78 haematopoietic progenitor cells
  • a number of routes for the production of the wild-type molecule as well as engineered variants with improved physico-chemical properties are described in Patent Application WO-A- 93/13206.
  • Macrophage Inflammatory Protein-1 has been recognised as a haemopoietic stem cell proliferation inhibitor by its capacity to protect multipotent progenitor cells from cytotoxic agents which are effective against cells in DNA synthesis, both in vitro and in vivo (1-7).
  • MIP-1 ⁇ was given in vivo to protect haemopoietic spleen colony-forming units (CFU-S) (8) from the effects of hydroxyurea (HU)
  • CFU-S haemopoietic spleen colony-forming units
  • stem cell inhibitor molecules such as MIP-1 ⁇ can be used to protect haematopoietic stem cells from cycle specific cytotoxic agents.
  • the present invention is based on the findings that continuous (as opposed to multiple bolus injections) administration of the SCI starting (a) before commencement of a course of myelosuppressive therapy or (b) during a course of myelosuppressive therapy or (c) immediately after a course of myelosuppressive therapy has ceased, and proceeding after the therapy has finished, has unexpected beneficial effects on the recovery of haematopoiesis.
  • continuous (as opposed to multiple bolus injections) administration of the SCI starting (a) before commencement of a course of myelosuppressive therapy or (b) during a course of myelosuppressive therapy or (c) immediately after a course of myelosuppressive therapy has ceased, and proceeding after the therapy has finished has unexpected beneficial effects on the recovery of haematopoiesis.
  • similar benefits are implied for the use of other SCCs in the same way as SCIs.
  • a method for enhancing haematopoietic recovery in a subject in need thereof which comprises commencing continuous administration of a stem cell chemokine to the subject
  • stem cell chemokine or “SCC” as used throughout this specification refers to any chemokine molecule or analogue or variant thereof to which haematopoietic stem and progenitor cells or their progeny respond. That definition includes any chemokine which has demonstrated biological activity on a haematopoietic stem cell, progenitor cell, maturing lineage specific cell, mature cell of either myelomonocytic or lymphoid lineages including but not limited to monocytes, macrophages, neutrophils, platelets, basophils, eosinophils, dendritic cells etc.
  • Chemokines or their analogues encompassed in this definition possess significant amino acid identity (>20% homology) or structural similarity to all or part of the family of chemotactic cytokines called chemokines, or if produced via recombinant DNA expression, the nucleic acid encoding the chemokine or analogue would hybridise under stringent conditions to nucleic acid encoding a known chemokine, such as LD78, or would do so but for the redundancy of the genetic code.
  • LD78 belongs to a super-family of related chemotactic cytokines (19). which have recently been called Chemokines. All of the chemokines have four conserved cysteines and are grouped into two sub-families according to their chromosomal location and the position of the first two cysteines, which are either adjacent (CC proteins or ⁇ subfamily; Iocated on human chromosome 4) or separated by one amino acid (CXC proteins or ⁇ subfamily; Iocated on human chromosome 17). Both ⁇ and ⁇ subfamilies of the chemokine superfamily are included in this definition.
  • chemokines share amino acid homology (the amino acid sequences of the chemokines can be found in various sequence databases such as EMBL or SwissProt) and have very similar tertiary structures. This means that information obtained for one is likely to be applicable to others. Baggiolini et al. (20) review the various members of the ⁇ and ⁇ subfamilies of the chemokine superfamily.
  • stem cell chemokines include but are not limited to: LD78 (huMIP-1 ⁇ ), muMIP-1 ⁇ , MIP-1 ⁇ (ACT- 2), Rantes, IL-8, GRO ⁇ , GRO ⁇ , GRO ⁇ , neutrophil activating protein (NAP-2), monocyte chem-attractant and activating protein (MCAF), epithelial cell-derived neutrophil activating protein (ENA78), platelet factor 4 (PF4), interferon-gamma inducible protein ( ⁇ lP10), granulocyte chemotactic protein 2 (GCP-2), MCP-1 , MCP-2 and MCP -3.
  • SCC molecules are known stem cell inhibitors, including: IL-8, PF4, MIP-1 ⁇ , Rantes, I N PRO L etc.
  • variant (or its synonym for present purposes “analogue”) is used, broadly, in a functional sense. Variants may possess amino acid deletions, substitutions and/or insertions. As a practical matter, though, most variants will have a high degree of homology with the prototype molecule if biological activity is to be substantially preserved. Variants or analogues may have improved biophysical properties and include those proteins capable of being more easily expressed or purified. Variants may also possess less toxicity when administered to the patient. It will be realised that the nature of changes from the prototype molecule is more important than the number of them.
  • nucleic acid coding for an analogue may for example hybridise under stringent conditions (such as at approximately 35°C to 65°C in a salt solution of approximately 0.9 molar) to nucleic acid coding for the prototype molecule, or would do so but for the degeneracy of the genetic code.
  • the stem cell chemokine for use in the invention is huMIP-1 ⁇ , in a more preferred embodiment it is BB-10010 a demultimerised LD78(huMIP-1 ⁇ ) analogue (as described in Example 7 of Patent Application No. WO- A-93/13206).
  • Myelosuppressive (also termed myeloablative) therapy refers to treatments that cause marrow and haematopoietic cells to be destroyed, but does not include their complete annihilation. Such treatments include chemotherapy and radiotherapy. After myelosuppressive therapy, the haematopoietic system is damaged and the levels of circulating mature blood cells, maturing lineage specific cells in haematopoietic tissues and stem cells are reduced.
  • haematopoietic recovery as used herein is meant the process of renormalisation of the haematopoietic system after such damage. The system may be said to have recovered completely when the levels of cells measured have risen to be within the range normally expected for each cell type.
  • CD34+ 1800-16200/ml blood CFU- GM 17-430/ml blood
  • BFU-E 20-190/ml blood white blood cells 3-11 x 10 9 /L
  • red blood cells 3.8 - 6 x 10 12 /L platelets 140 - 400 x 10 L
  • neutrophils 1.7 - 7.5 x 10 9 /L lymphocytes 1 - 3.5 x 10 9 /L
  • procedures for estimating cell numbers are interoperator and interlaboratory specific, with interassay variation often resulting in different estimations of cell numbers.
  • the rate of haematopoietic recovery (which is faster when the method of the invention is used than with the methods of the prior art) can be estimated by use of assays known in the art of murine haematopoiesis. These include the marrow repopulating ability (MRA) assay (also known as the pre-CFU-S assay), the CFU-S assay, nucleated cell counts from femoral cell suspensions or from blood and red cell counts from these tissues. These assays are described in: Haematopoiesis, A Practical Approach, Testa NG, Molineux G (eds):Oxford,New York,Tokyo, IRL Press at Oxford University Press, 1993.
  • MRA marrow repopulating ability
  • assays In humans, not all of these assays are possible. However, a plethora of appropriate assays are known which can be used to estimate the state of haematopoiesis at steady state and after myeloabiative insult or therapy. Any of these methods could be used singly or in combination to estimate the state of haematopoietic recovery.
  • These assays are clonogenic in vitro assays eg. CFU-GM, BFU-E, CFU-GEMM etc, LTCIC and CAFC assays (see ref:18 and 21-23), marrow cellularity and mature cell number counts including neutrophils, platelets or lymphocytes, taken from marrow biopsies or other haematopoietic tissues or from blood.
  • the benefit of the invention is observed if a faster recovery to a certain value (eg. time to neutrophil count >500/mm3) or greater number at a certain time (eg. number of CFU-GM or LTCIC at 10 days post myelosuppressive therapy) of any of the haematopoietic cell types is measured compared to control value without any stem cell chemokine treatment or by treatment using the regimes of the prior art.
  • the benefit of the invention is also observed if no changes in the rate or extent of recovery were seen but if individuals which received stem cell chemokine treatment in accordance with the invention are better able to withstand subsequent rounds of myelosuppressive insult or therapy. This is seen as faster or greater recovery in subsequent rounds of therapy, or by a reduction in the level of toxicity in subsequent rounds of repeated cycles of chemotherapy.
  • Faster mature cell recovery may be an indication of the ability to withstand subsequent rounds of chemotherapy or radiotherapy, however it should not be the sole guide.
  • the state of the marrow is a far better guide. Greater numbers of early progenitors or stem cells gives a better indication of the "quality" of the marrow recovery. If these levels approach the numbers found in normal individuals then the marrow is more fully recovered and is more likely to be able to sustain haematopoiesis after subsequent rounds of therapy.
  • This invention calls for continuous stem cell chemokine treatment over a prolonged period. This means that the prolonged nature of its use may indeed inhibit the recovery of the mature cells whilst improving the quality of the marrow stem and progenitor cell recovery.
  • stem cell effects may occur at the expense of mature cell recovery.
  • the positive effects of prolonged continuous administration of the stem cell chemokine on stem cell recovery may be at the price of slower mature cell recovery, and this may therefore require therapeutic intervention with growth factors or by transfusions of neutrophils, platelets or blood to sustain the patient through the nadir of myelosuppression.
  • the beneficial effects of the stem cell chemokine may become apparent in subsequent rounds of myelosuppressive therapy when the greater number of stem cells will allow the marrow to withstand the myelosuppression even better.
  • the clinician may choose to balance the optimum effects of the stem cell chemokine on recovery with the potential prolonged cytopenia.
  • the ability to undergo more rounds of myelosuppressive therapy is of great clinical significance. Often patients do not recover from the first few rounds of therapy and cannot proceed to the later rounds in a multiple cycle regimen. This reduces the rate of remission and the immunocompromised patients are more susceptible to opportunistic infection. It would be much better if a greater proportion of patients could continue all of the planned rounds of therapy and if restoration of the immune system was speeded up.
  • the use of this invention increases the ability of patients to withstand additionai rounds of therapy and/or higher dosages of the myelosuppressive insult than currently possible.
  • the invention is applicable following cycle specific and non-cycle specific chemotherapies and radiotherapy.
  • the invention provides for enhanced haematopoietic recovery after myeloabiative therapy by prolonged continuous administration of a stem cell chemokine
  • a pharmaceutical composition for continuous administration comprising a stem cell chemokine.
  • continuous administration means a mode of administration whereby the agent is administered to the subject such that the agent is maintained in the body, preferably the blood, at substantially the same concentration over time, as opposed to administrations that result in a peak in the concentration of the agent followed by a gradual or rapid decrease in concentration, eg. following bolus intra venous injection.
  • Such administration may comprise continuous infusion.
  • continuous does not imply that uninterrupted administration, or administration for 24 hours per day, is essential.
  • Continuous administration of the stem cell chemokine is generally via a device which ensures delivery for a substantial proportion of each day of treatment.
  • the continuous administration should be for more than 4hr, 8hr, 12hr, 16hr, 20hr or 23hr.
  • the SCC is administered continuously throughout each day without interruption.
  • delivery devices may need to be replenished and that there may be periods when administration is discontinuous as described above.
  • patients may require other therapies during the course of their treatment which may be related to the use of stem cell chemokines, the patients underlying disease state or for entirely different reasons which are incompatible with stem cell chemokine administration.
  • the stem cell chemokine administration could be suspended until it was appropriate to resume administration.
  • Continuous administration can be via i.v., i.p., i.m. or s.c. routes. Other routes such as transdermal may also be possible.
  • the ablation of the haematopoietic system caused by myelosuppressive therapies is not instantaneous but takes several days for the nadir of cytopenia to develop.
  • Cytotoxic chemotherapeutic myelosuppressive agents once injected have a clearance ⁇ -half-life from the body which is agent specific eg. 5-FU approx 10min, AraC 2hrs, Cyclophosphamide 10hrs.
  • agent specific eg. 5-FU approx 10min, AraC 2hrs, Cyclophosphamide 10hrs.
  • the clinician may decide to begin administration for a period of up to 7 days before the myelosuppressive treatment is initiated.
  • stem cell chemokine or stem cell inhibitor
  • the stem cell chemokine (or stem cell inhibitor) treatment would normally be continued throughout the duration of the myelosuppressive treatment.
  • the myeloabiative treatment would be completed within 24hrs although longer treatment regimens would be considered.
  • the clinician would then seek to continue the protection with stem cell chemokine until the body had cleared sufficient of the cytotoxic chemotherapeutic myelosuppressive agent to render any remaining agent ineffective. A clinician may decide that 50%, 25%, 12.5% or 6.25% of remain drug might be an ineffective dose.
  • the stem cell chemokine should not be administered throughout the duration of the multiple cycles of chemotherapy. There should preferably be a break in treatment with stem cell chemokine before the next cycle of myelosuppressive treatment begins. This break should preferably be >25% and not less than 10% of the cycle duration.
  • the stem ceil chemokine may be administered for an undefined period of time before myelosuppressive treatment begins, throughout the day of the treatment (day 0)and preferably for at least 8 ⁇ half-lives of the myelosuppressive agent after administration of the agent ceases. If a cocktail of myelosuppressive agents has been used then the longest ⁇ half-life should be used in the calculation. For AraC this period would be 64hr after AraC treatment ceased or through days 1 and 2 and part way through day 3 of the clinical protocol. If beneficial the duration of administration of stem cell chemokine could be even longer. The maximum duration of administration is for 90% of cycle time but preferably 75% or less of cycle time.
  • this invention calls for continuous administration of stem cell chemokine for a minimum of days 0, 1 and 2 and part of day 3 of the experimental protocol and not longer than from day 0 through to day 26 and preferably no longer than day 22 of the clinical protocol.
  • Clinical regimens which administer a stem cell chemokine for a shorter period that 8 ⁇ half-lives of the myelosuppressive agent after the agent has ceased to be administered are not the subject of this invention.
  • stem cell chemokine dosage regimens seek only to use the stem cell inhibitory and therefore protective effects of some stem cell chemokines such as LD78 and do not attempt to make use of the utility of prolonged use of stem cell chemokines on self-renewal or regeneration of the haematopoiesis system.
  • the invention aiso includes the use of a stem cell chemokine in the preparation of a medicament for use in continuous administration for:
  • a method for enhancing haematopoietic recovery in a subject having undergone, or undergoing myelosuppressive therapy comprises the continuous administration of an effective amount of a stem cell chemokine.
  • An element to haematopoietic recovery arising from the effects of continuous administration of a SCC is the enhanced mobilisation of haematopoietic cells to the blood.
  • stem cell chemokine for use in continuous administration is LD78 or MIP-1 ⁇ or analogues thereof.
  • the de-multimerised LD78 analogue BB-10010 (as described in Patent Application No. WO-A-93/13206) is used in continuous administration.
  • Molecules useful in the present invention can be prepared from natural or recombinant sources. Methods for expression and purification of stem cell chemokines by recombinant techniques are known in the art.
  • the preferred form of the natural MIP-1 ⁇ molecule is a 69 amino acid form of LD78 described by Obaru e al. (25).
  • the active ingredient may be administered parenterally in a sterile medium.
  • the drug can either be suspended or dissolved in the vehicle.
  • the stem cell chemokine will be administered in the form of a sterile composition comprising the purified protein in conjunction with physiologically acceptable carriers, excipients or diluent.
  • adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.
  • suitable pharmacological compositions is routine to those skilled in the art, as exemplified by "Remington's Pharmaceutical Sciences” 15th Edition, incorporated herein by reference.
  • Dosage of stem cell chemokines in accordance with any aspect of the invention will be such as to be effective and will be under the control of the physician or clinician considering various factors such as the condition, sex, body weight and diet of the patient and the severity of the myelosuppressive treatment administered.
  • doses may be in the range of from 0.1 ⁇ g/kg/day and 10mg/kg/day, preferably a dose between 1 ⁇ g/kg/day and 1 mg/kg/day more preferably from 10 ⁇ g/kg/day to 0.2 mg/kg/day.
  • the most effective dose of stem cell chemokine given throughout the treatment can be determined by clinicians following normal clinical research practice in dose-response studies.
  • the optimal dose may be constant throughout the duration of dosing or may change. For instance a high dose initial induction might be appropriate.
  • the invention will be described in the following non-limiting examples and by the following figures in which:
  • Figures 1 and 2 diagrammatically represents the data of Tables 3 and 4 of eight and twelve day CFU-S in the bone marrow of mice subjected to repeated 14 d cycles of 4.5 Gy y -rays.
  • the graphs refer to mini-osmotic pump dispensing BB-10010 (•) or PBS (O).
  • Figures 3 and 4 diagrammatically represents the data of Tables 3 and 4 of eight and twelve day CFU-S in the spleen of mice subjected to repeated 14 d cycles of 4.5 Gy y -rays.
  • the graphs refer to mini-osmotic pump dispensing BB-10010 (•) or PBS (O).
  • Figures 5 and 6 diagrammatically represents the data of Tables 3 and 4 of the 1 day post irradiation nadirs in bone marrow CFU-S following sequential 2- weekly doses of 4.5 Gy ⁇ -rays.
  • O Irradiation only;
  • Figures 7 and 8 diagrammatically represents the data Tables 3 and 4 of the 14 day post irradiation recovery values for bone marrow CFU-S following sequential 2-weekly doses of 4.5 Gy ⁇ -rays.
  • mice Male B6D2F1 (C57BI? x DBA2 ) mice aged 10 wks at the start of experiments were used throughout and all procedures were carried out under licence from the Home Office, Animals (Scientific Procedures) Act, 1986. BB-10010/MIP-1 ⁇
  • BB-10010 is a non-aggregating, genetically engineered variant of human MIP-1 ⁇ (or LD78) comprising a single amino acid substitution of Asp26>Ala.
  • the construction of BB-10010 is described in example 7 in WO-A-9313206.
  • mice (groups of 20) were irradiated with 15.25 Gy 60Co ⁇ -rays (0.95 Gy/hr). They were then injected intravenously with 0.2 ml of a freshly prepared suspension of bone marrow or spleen cells from mice treated as described below. Eight days (10 mice) and 12 days (10 mice) later the recipient mice were killed. Their spleens were excised, fixed and the colonies counted.
  • MRA Marrow Repopulating Ability Assays
  • the MRA was measured as the generation of 12 d CFU-S during 13 days' growth in the marrow by an extension of the CFU-S assay (see ref. 13 for details), on day 14 of the 3rd and 4th treatment cycles.
  • An extra 5 irradiated (primary) recipients were injected with the bone marrow suspension. After 13 days their femora were removed. Bone marrow suspensions were made and assayed for CFU-S12 in secondary groups of 10 irradiated recipients.
  • MRA was calculated as (c x p x q)/N per femur or c x p x q x IO 5 per IO 5 cells:
  • 1/q fraction of donor marrow cells injected into primary recipient
  • 1/p fraction of primary recipient marrow injected into secondary recipient.
  • mice Groups of 3 mice were implanted subcutaneously with mini-osmotic pumps delivering BB-10010 (40 ⁇ g/24 hr period for 7d) or Phosphate Buffered Saline (PBS). Three to four hours later the mice were exposed to 4.5 Gy ⁇ -rays from a caesium-137 source (dose rate 2.5 Gy/min) and after 7 days the spent pumps were removed. Groups of mice were killed at 1 , 7 and 14 days after irradiation and their femora and spleens removed. Cell suspensions were made in Fischer's medium from the bone marrow and spleen (see ref.
  • BM bone marrow
  • Iabje_2 Cellularity of Bone Marrow and Spleen in Mice Subjected to Repeated Cycles of Sub-Lethal Irradiation. With or Without
  • mice were exposed to 4.5 Gy ⁇ -rays on day 0 and at 14 day intervals thereafter. Data are for 3 to 4 experiments ⁇ standard error. Data for day 0 are standardized norms for these mice and are presented simply as approximate reference points.
  • T able 3 8 d_CFU ⁇ S in Bone Marrow and Spleen of Mice Subjected to Repeated Cycles of Sub-Lethal lnadia.tjojL.Wjth gr .Without BB-10010.
  • mice were exposed to 4 5 Gy ⁇ -rays on day 0 and at 14 day intervals thereafter. Data are for 3 to 4 experiments 1 standard error. Data for day 0 are standardized norms for these mice and are presented simply as approximate reference points.
  • mice were exposed to 4.5 Gy ⁇ -rays on day 0 and at 14 day intervals thereafter. Data are for 3 to 4 experiments 1 standard error. Data for day 0 are standardized norms for these mice and are presented simply as approximate reference points.
  • e 3_ M A of Progenitor Cells in Bone Marrow of Mice Subjected to Repeated Cycles of Sublethal Irradiation, With or Without
  • Marrow repopulating ability is normally recorded for these mice at about 10 s per femur or 500 per 10 5 marrow cells (14).
  • Fourteen day recovery-marrow in the 3rd and 4th cycles of sublethal irradiation yielded only 6500-7300 per femur which is less than 10% of normal (Table 5).
  • Treatment with BB-10010 yielded an MRA of 26600 after the third cycle and 11206 after the fourth cycle of irradiation (Table 5).
  • the cellular concentration of MRA cells was increase by 1.3-3 times following continuous BB-10010 administration treatment.
  • BB-10010 gave little, protection from the first dose of irradiation but the better recovery characteristics, particularly in respect of the highly enriched MRA, ensured that the second and subsequent irradiations caused progressively less initial damage and that the recovery patterns did not significantly deteriorate.
  • Figures 1 to 4, 7 and 8 show that CFU-S recoveries are generally much better with BB-10010 treatment.
  • Example 1 shows the ability of BB-10010 to improve haematopoietic recovery when it is continuously administered via minipump for 7 days. This example shows that a shorter period of continuous administration is much less effective in inducing this recovery.
  • Prolonged continuous administration of BB--10010 is more efficacious than multiple injections.
  • Example 1 and comparative example 1 show that prolonged continuous administration of BB-10010 for 7 days significantly improves haematopoietic recovery through multiple cycles of myelosuppressive therapy and that it is advisable to administer the BB-10010 for more than 3 days in this model system.
  • Comparative example 2 illustrates the value of continuous minipump administration of BB-10010 relative to repeated injections over the same period.
  • the methods were as in example 1 and comparative example 1 , using 2 cycles of radiation treatment together with BB-10010 treatment over the first 7 days of each cycle.
  • BB-10010 was administered by (a) 7 day minipump (40 ⁇ g/d), (b) by daily subcutaneous injections of 40 ⁇ g or (c) by twice daily injections of 20 ⁇ g.
  • the pumps were implanted 3 to 4hr and the injections started 2 hrs before irradiation.
  • CFU-S were assayed after 14 days recovery in the second treatment cycle. Table 7 show that only continuous administration by pump provided significant enhancement in CFU-S recovery and illustrates the advantage of this form of administration relative to a repeated bolus injection regimen.
  • BB-10010 administration was started 3 to 4 hours prior to the radiation treatment. It is possible that BB-10010 protected stem cells from the myelosuppressive therapy as well as, or instead of, enhancing their recovery. Examples 1 and comparative example 1 provide evidence which argues that much of the haematopoietic enhancing effect of prolonged continuous BB-10010 administration may occur after several days of BB-10010 administration post-irradiation.
  • the 3 and 7 day minipump experiments had the same pre-irradiation dosing of BB-10010 so their direct protective effects should be identical; but the 7 day minipumps were considerably more effective than the 3 day minipumps. This suggests that the additional 4 days of administration are important to the ultimate enhancing effects of BB-10010.
  • this example describes the activity of BB-10010 dosed continuously for 7 days via minipumps inserted after the cessation of radiation. In this study there can be no direct protective contribution of BB-10010.
  • Minipumps implanted before irradiation (0-7d) generated good enhancement of recovery giving twice as many CFU-S/f. Pumps fitted 1 day after irradiation showed less efficacy (-1.4 fold enhancement) while those present in the second phase of the recovery cycles were ineffective.
  • BB-10010 can protect the progenitor CFU- S population from a generalised myelosuppressive agent (eg radiation) while its subsequent, but not delayed, continuous availability is separately instrumental in promoting the recovery rate of the surviving CFU-S.
  • a generalised myelosuppressive agent eg radiation
  • BB-10010 Continuous administration of BB-10010 enhances the peripheral blood mobilisation of progenitor cells from the bone marrow.
  • BB-10010 can enhance the efficacy of progenitor cell mobilisation from marrow to peripheral blood induced by G-CSF (24).
  • Prolonged continuous administration of BB-10010 improves the efficacy of progenitor cell mobilisation bv G-CSF.
  • Hendry JH, Lajtha LG The response of hemopoietic colony-forming units to repeated doses of X-rays. Radiat Res 52: 309, 1972.
  • Ploemacher RE, van Os RP, van Buerden CAJ, Down JD Murine hemopoietic stem cells with long-term engraftment and marrow repopulating ability are less radiosensitive to gamma irradiation than are spleen colony forming cells. Int J Radiat Biol 61 : 489, 1992.

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Abstract

La présente invention, qui concerne une amélioration de la reprise de l'hématopoïèse à la suite d'une thérapie myélosuppressive, consiste en l'administration continue d'une chémokine (ou cytokine chimiotactique) de cellules souches.
PCT/GB1996/002006 1995-08-19 1996-08-19 Reprise de l'hematopoiese WO1997006817A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU67501/96A AU6750196A (en) 1995-08-19 1996-08-19 Haematopoietic recovery

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9517014.8 1995-08-19
GB9517014A GB9517014D0 (en) 1995-08-19 1995-08-19 Haematopoietic recovery

Publications (1)

Publication Number Publication Date
WO1997006817A1 true WO1997006817A1 (fr) 1997-02-27

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Family Applications (1)

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PCT/GB1996/002006 WO1997006817A1 (fr) 1995-08-19 1996-08-19 Reprise de l'hematopoiese

Country Status (3)

Country Link
AU (1) AU6750196A (fr)
GB (1) GB9517014D0 (fr)
WO (1) WO1997006817A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0848012A1 (fr) * 1996-12-13 1998-06-17 Roche Diagnostics GmbH Utilisation de polypeptides pour le traitement de la thrombocytopénie
EP0866806A4 (fr) * 1995-10-24 2002-01-09 Smithkline Beecham Corp Procede de mobilisation de cellules souches hematopoietiques

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993013206A1 (fr) * 1991-12-23 1993-07-08 British Bio-Technology Limited Proteines inhibant les cellules souches
WO1996019234A1 (fr) * 1994-12-22 1996-06-27 British Biotech Pharmaceuticals Limited Mobilisation de cellules hematopoietiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993013206A1 (fr) * 1991-12-23 1993-07-08 British Bio-Technology Limited Proteines inhibant les cellules souches
WO1996019234A1 (fr) * 1994-12-22 1996-06-27 British Biotech Pharmaceuticals Limited Mobilisation de cellules hematopoietiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
B. I. LORD ET AL.: "MACROPHAGE INFLAMMATORY PROTEIN: ITS CHARACTERISTICS, BIOLOGICAL PROPERTIES AND ROLE IN THE REGULATION OF HAEMOPOIESIS.", INTERNATIONAL JOURNAL OF HEMATOLOGY, vol. 57, no. 3, June 1993 (1993-06-01), pages 197 - 206, XP000611694 *
M. G. HUNTER ET AL.: "BB-10010: AN ACTIVE VARIANT OF HUMAN MACROPHAGE INFLAMMATORY PROTEIN-1 ALPHA WITH IMPROVED PHARMACEUTICAL PROPERTIES.", BLOOD, vol. 86, no. 12, 15 December 1995 (1995-12-15), pages 4400 - 4408, XP000611695 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0866806A4 (fr) * 1995-10-24 2002-01-09 Smithkline Beecham Corp Procede de mobilisation de cellules souches hematopoietiques
EP0848012A1 (fr) * 1996-12-13 1998-06-17 Roche Diagnostics GmbH Utilisation de polypeptides pour le traitement de la thrombocytopénie
WO1998025966A1 (fr) * 1996-12-13 1998-06-18 Roche Diagnostics Gmbh Utilisation de polypeptides pour le traitement de la thrombocytopenie
US6403553B1 (en) 1996-12-13 2002-06-11 Wolf-Georg Forssmann Use of polypeptides for treating thrombocytopenia

Also Published As

Publication number Publication date
GB9517014D0 (en) 1995-10-25
AU6750196A (en) 1997-03-12

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